Summary In the last grant period, we focused on the determinants of the selective vulnerability of substantia nigra (SN) dopaminergic (DA) neurons in Parkinson's disease (PD), building out the scientific foundation for the ongoing NINDS-sponsored, Phase 3 clinical trial with isradipine. These studies and those of others in the field clearly point to mitochondrial dysfunction as a key factor in PD pathogenesis. Our plan is to pursue this connection in a novel mouse model of PD pathogenesis that results from conditional deletion of the Ndufs2 subunit of mitochondrial complex I (MitoCI). These mice, which we refer to as MitoCI-Park mice, manifest a progressive, levodopa-responsive parkinsonism. We propose to use a battery of cutting-edge optical, electrophysiological, electrochemical, and transcriptomic tools to achieve three specific aims. Our first aim is to characterize the mechanisms driving the progressive dysfunction of SN DA neurons in presymptomatic MCI-Park mice. In this period, axonal release of DA declines and roughly half of the SN DA neurons lose their phenotype. Our working hypothesis is that deficits in axonal bioenergetics and retrograde mitochondrial signaling drive this progressive loss of function. Our second aim is to characterize the mechanisms driving the progressive loss of SN DA neurons in symptomatic MitoCI-Park mice. Our working hypothesis is that in addition to the mechanisms operating in the pre-symptomatic period, network dysfunction and activation of metabotropic glutamate receptors contribute to the pathology in this period. Our third aim is to determine whether parkinsonism in MitoCI-Park mice can be reversed by restoring mitochondrial function. Our working hypothesis is that the parkinsonian state in the MitoCI-Park mouse is driven initially by the down- regulation in the dopaminergic phenotype, not frank neuronal loss, and restoring MitoCI function will restore the phenotype and behavior. These aims will be pursued in close collaboration with Projects 2 and 3. Linkage to other projects: Project 2 focus on a very similar set of questions in the MitoCI-Park mouse using a combination of in vivo and ex vivo network approaches that fully complement the more cellular approaches used here. Project 3 is focused on cell autonomous factors consequences of MitoCI dysfunction in human DA neurons, allowing not only species comparisons but also clear distinctions between cell autonomous and network mechanisms. Also, both Project 2 and 3 will pursue questions related to DA release, a major focus of this project; both projects will attempt Mito-CI rescue using the methods developed here. Thus, all three projects are tightly interconnected and will inform and cross-check the conclusions drawn from each set of experiments.